Team:Berkeley/Methods

Purpose:

The Berkeley iGEM team 2013 has determined the enzyme kinetics of FMO and GLU (data for: FMO and GLU) based on the Michaelis-Menten kinetics model. Our purpose of determining kinetics was to give a quantitative data of how fast these enzymes can turnover their respective substrates, which is very useful when considering scaling up the process and for future iGEM teams when they use our enzymes.

Materials:

• Purified Enzyme: FMO or GLU
• Substrate:
  • FMO: Indole, NADPH (co-factor)
  • GLU: Indican
• TECAN Plate Reader and TECAN Plate
• Solvents: DMSO, Water

Methods:

Note: We followed the protocol from Bio-Rad’s Bradford Assay to determine the concentration of purified FMO and GLU.

We determined the concentration of our purified enzyme to be 4.2 mg/L for FMO and 3.5 mg/L for GLU.

Note: We assumed that the oxidation of indoxyl to indigo is much faster than that of the turnover rate of indole to indoxyl by FMO.

1. 1. In each well of the TECAN plate, we added 0.3 mM of NADPH, purified FMO and various indole concentrations (0, 0.10, 0.19, 0.39, 0.78, 1.55, 3.10, 6.20, 12.40 mM) at 5% DMSO.

Note: The 5% DMSO originates from the fact that indole had to be dissolved in DMSO. We recommend minimal DMSO because FMO is sensitive to DMSO.

2. 2. Using a TECAN plate reader, we determined the absorbance at 620 nm (peak wavelength at which indigo absorbs) every minute for 50 minutes.
3. 3. The absorbance data was converted into concentration of indigo based on the indigo standard we have determined previously.
4. 4. We wrote the following MATLAB code to automate analysis and generated the Michaelis-Menten Km.